Exposure of cells to ionizing radiation and other carcinogenic agents induces DNA double-strand breaks (DSBs). Genetic studies in Saccharomyces cerevisiae and other organisms have revealed that homologous recombination (HR) and non-homologous DNA end joining (NHEJ) represent the major mechanisms for DSB elimination. During recombinational repair, ssDNA tails derived from break processing are bound by HR factors. The nucleoprotein complex thus formed conducts a search to locate an undamaged DNA homolog and catalyzes the formation of heteroduplex DNA joints with the homolog. The RAD50, RAD51, RAD52, RAD55, RAD57, RAD59, MRE11, and XRS2 genes are key members of the evolutionarily conserved RAD52 epistasis group that mediate mitotic and meiotic recombination and the recombinational repair of DSBs. The proteins encoded by MRE11, RAD50, and XRS2 are associated in a complex that plays roles in both HR and NHEJ. Rad51 protein, with the aid of ancillary factors, nucleates onto the ssDNA tails to form a nucleoprotein filament that has the ability to initiate heteroduplex DNA formation. In our renewal project, a combination of biochemical and genetic approaches will be used to (i) delineate the functions of MRE11/Rad50/Xrs2 in DSB repair, (ii) continue dissecting the mechanism of action of the Rad51-associated complex in heteroduplex DNA formation and chromatin remodeling, and (iii) define the multifaceted role of the Srs2 helicase in HR and DSB repair. Our studies will be important for deciphering the mechanism and functional significance of DSB repair in eukaryotes. Since defective DSB repair causes several human cancer-prone syndromes, the results from our project will be highly germane for understanding the biology and etiology of radiation-induced and chemical-induced carcinogenesis.